DESCRIPTION: (applicant's abstract) The enzyme guanylate cyclase catalyzes the formation of cGMP from GTP and exists in both soluble and particulate fractions of cells. The soluble form of the enzyme (sGC) is a heme-containing heterodimer whose activity can be up-regulated by heme-dependent ligands such as nitrovasodilators and free radicals such as nitric oxide as well as heme-independent ligands such as arachidonic acid and protoporphyrin IX. The mechanism of activation of the enzyme by either type of ligand is as yet unknown. In vivo sGC is a cellular receptor for nitric oxide (NO) and takes part in transducing the NO signal to down stream effectors such as protein kinases, phosphodiesterases and ion channels. These downstream effectors mediate the physiological effects of NO. The involvement of NO in vascular smooth muscle relaxation, platelet aggregation and neuronal potentiation and plasticity have been linked to its activation of sGC. The goal of the proposed research is to further define the structural requirements of the heme prosthetic group and activating ligands for the up-regulation of sGC. In addition, the correlation between activation and conformational changes in the protein will be studied in an effort to determine whether a common mechanism for activation exists between heme-dependent and heme-independent ligands. The effects of such heme dependent ligands as NO, CO, and CN, and heme independent ligands such as arachidonic acid and protoporphyrin IX will be studied. This project will utilize a baculovirus expression system to produce sGC protein using cDNAs from lung and brain tissues. Effects of ligand binding to the heme will be monitored by UV-Visible spectroscopy and the nature of the heme pocket examined by FT-IR spectroscopy. Conformational changes in the protein due to ligand binding will be studied using circular dichroism (CD) and florescence spectroscopy. This study will provide important information about the structural requirements for activation of this biologically important enzyme. It will allow a model to be built that can be used for future studies on the role of the protein structure in the activation process. The information gained from this study will help further the design of inhibitors of the enzyme which, given the role that sGC plays in the biology of nitric oxide, is a critical step in developing treatments of NO related pathologies.